Polyphenols from the Peels of Punica granatum L. and Their Bioactivity of Suppressing Lipopolysaccharide-Stimulated Inflammatory Cytokines and Mediators in RAW 264.7 Cells via Activating p38 MAPK and NF-κB Signaling Pathways

Punica granatum L. (Punicaceae) is a popular fruit all over the world. Owning to its enriched polyphenols, P. granatum has been widely used in treating inflammation-related diseases, such as cardiovascular diseases and cancer. Twenty polyphenols, containing nine unreported ones, named punicagranins A–I (1–9), along with eleven known isolates (10–20), were obtained from the peels. Their detailed structures were elucidated based on UV, IR, NMR, MS, optical rotation, ECD analyses and chemical evidence. The potential anti-inflammatory activities of all polyphenols were examined on a lipopolysaccharide (LPS)-induced inflammatory macrophages model, which indicated that enhancing nitric oxide (NO) production in response to inflammation stimulated in RAW 264.7 cells was controlled by compounds 1, 3, 5–8, 10, 11, 14 and 16–20 in a concentration-dependent manner. The investigation of structure–activity relationships for tannins 6–8 and 12–20 suggested that HHDP, flavogallonyl and/or gallagyl were key groups for NO production inhibitory activity. Western blotting indicated that compounds 6–8 could down-regulate the phosphorylation levels of proteins p38 MAPK, IKKα/β, IκBα and NF-κB p65 as well as inhibit the levels of inflammation-related cytokines and mediators, such as IL-6, TNF-α, iNOS and COX-2, at the concentration of 30 μM. In conclusion, polyphenols are proposed to be the potential anti-inflammatory active ingredients in P. granatum peels, and their molecular mechanism is likely related to the regulation of the p38 MAPK and NF-κB signaling pathways.

Punicagranin D (4) was a pale yellow powder with molecular formula of C 15 H 12 O 9 deduced from HRESIMS analysis. The thin layer chromatography (TLC) of it showed bluepurple fluorescent spots under the UV lamp of 365 nm before and after spraying with 10% aqueous H 2 SO 4 -EtOH. Additionally, its UV spectrum displayed the maximum absorption peaks at 274 and 344 nm. Then, punicagranin D (4) was speculated to be a coumarin.
Finally, its Cotton effects displayed at 326 nm (positive), 298 nm (negative) and 255 nm (negative) were consistent with those of methyl (S)-heptamethylflavogallonate [24], suggesting the chirality of the flavogallonyl group was in the S-series. Moreover, its opposite [25] further clarified the conclusion. Then, the structure of punicagranin F (6) was elucidated to be ethyl (S)-flavogallonate. Though the plannar structure of it had been speculated by Abiodun et al. through LC-MS analysis [26], the absolute configuration of it was expounded here for the first time.
Punicagranin G (7)  However, sixty-eight signals displayed in its 13 C NMR spectrum. The 1 H and 13 C NMR spectra of 7 showed duplicated signals for the sugar and polyphenol moieties, indicating an equilibrium mixture of α and β anomeric forms with the ratio about 1:2 in solution. After hydrolysis with 1 M HCl, D-glucose was detected from its hydrolysate [27]. The cross-peaks between δ H 2.46 (1H, dd, J = 3.6, 9.6 Hz, Glc-H-2α) and δ D-glucosyl disappeared, and D-fructofuranosyl appeared in compound 8, which was clarified by the cross peaks showed in the 1 H 1 H COSY spectrum, as well as the correlations from H 2 -1 to C-2, C-3 displayed in its HMBC spectrum ( Figure 2). The correlations from H-4 to C-7 ; H 2 -6 to C-7 and the ECD similarity between it and compound 7 suggested the structure of punicagranin H (8) was 4,6-(S,S)-gallagyl-D-fructofuranose. It was a mixture of 4,6-(S,S)-gallagyl-α-D-fructofuranose and 4,6-(S,S)-gallagyl-β-D-fructofuranose with the ratio 1:3 nearly.
As a result, D-glucose was detected [27]. The 1 H, 13  The structures of the known compounds 10-20 were identified by comparing the spectroscopic data with those reported in literature. Among all of the obtained compounds, 6-8 and 12-20 were tannins.
To clarify whether compounds 2, 4-6 and 9 were artificial products, a 70% ethanol extract and 70% methanol extract of P. granatum peels, together with the reference compounds of 2, 4-6 and 9 were analyzed by LC-MS ( Figure S65, Supplementary Materials). As compounds 6 and 9 could not be detected in the 70% MeOH and 70% EtOH extracts, respectively, they were elucidated to be artificial products. Compound 6 might be produced by refluxing with ethanol in the process of P. granatum peel extraction. Compound 9 might be the reaction product of the sample and methanol used as the mobile phase.

In Vitro Anti-Inflammatory Evaluation of Compounds 1-20
As an excess of NO can trigger severe damage at the inflamed sites by amplifying the inflammation response [4] and exploiting new strategies to treat the over production; thus, the release of NO is important and urgent. The LPS-induced RAW 264.7 macrophages model was established to evaluate the potential anti-inflammatory activities of the twenty polyphenols (1-20) by measuring the NO level as what has been reported [29].
In order to test the NO release inhibitory effects of all compounds at a safe concentration, a MTT assay was conducted first. The results showed that at the concentration of 30 µM, all compounds showed no cytotoxic on RAW 264.7 cell, except compound 19, which showed no toxic until 15 µM ( Figure S72, Supplementary Materials). Thus, in the in vitro anti-inflammatory assay, the concentration for compounds 1-18, 20 and 19 were determined to be 30 µM and 15 µM, respectively. The results were compounds 1, 3, 5-8, 10, 11, 14 and 16-20 displayed significant inhibitory effects on the NO release levels in LPS-activated RAW 264.7 cells (Table 1) at a non-toxic concentration. It was found that compounds 1, 3, 5-8, 10, 11, 14, 16-18 and 20 reduced NO production in a concentration-dependent manner at 3, 10 and 30 µM, while compound 19 showed a similar mode at 1, 5 and 15 µM (Figure 3).
By comparing their bioactivities, the structure-activity relationships (SARs) can be summarized: (1) Tannins containing gallagyl (6-8 and 18-20) had good inhibitory activity on NO release production. Compound 19 displayed stronger activity than 17. Both of the results suggested gallagyl group may be a positive factor for NO inhibitory activity. (2) The HHDP group in tannins also played an important role in inhibiting NO release (18 vs . 19). (4) Moreover, it was found that ethyl or methyl esterification of carboxyl group could affect the activity slightly only (2 vs. 1 and 6 vs. 14). (5) Ellaganosides (12 and 13) showed no activity, which might be related to the introduction of sugar groups into ellagic acid (Table 1 and Figure 3).
This summary may provide more evidence for the structural modification of this type of polyphenols in the development of potential anti-inflammatory drugs.  respectively. Values represent the mean ± SD of six determinations. * p < 0.05, ** p < 0.01, *** p < 0.001 (Differences between the compound-treated group and control group). ### p < 0.001 (Differences between the control group and normal group).

Polyphenols from P. granatum Peels Exerted Anti-Inflammatory Effects through p38 MAPK and NF-κB Signaling Pathway
Moreover, the anti-inflammatory mechanism of compounds 6-8 at a concentration of 30 µM in LPS-induced RAW 264.7 macrophages model was investigated by western blot analyses. Comparing with the normal group, increased p38 mitogen-activated protein kinase (MAPK), IκB kinase (IKK)α/β, inhibitory nuclear factor κB-α (IκBα) and NF-κB p65 phosphorylation were observed in the model group.
Then, it was found that compounds 6-8 could down-regulate the phosphorylation levels of above-mentioned proteins. The expression levels of inflammation-related cytokines and mediators, such as IL-6, TNF-α, iNOS and COX-2 were down-regulated compared with the model group ( Figure 4). The obtained findings also revealed that compound 8 owned the best anti-inflammatory activities, either in inhibiting NO production or iNOS expression, as well as down-regulating the IL-6 level in the LPS-treated RAW 264.7 cells.  p38 MAPK is one major member of MAPKs. As upstream modulators of inflammatory molecules, MAPKs have been considered to play critical role in the regulation of inflammatory mediators and cytokines, such as COX-2, inducible nitric oxide synthase (iNOS) in macrophage cells [30]. The p38 MAPK pathway, plays an important role in inflammation in particularly [31].
According to the above-mentioned evidence, compounds 6-8 were confirmed to conduct anti-inflammatory activities by inhibiting p38 MAPK and NF-κB signaling pathways.
Combined with our investigation, polyphenols are proposed to be the potential antiinflammatory active ingredients in P. granatum peels.

Plant Material
The medicinal herbs of the dried P. granatum peels were purchased from Beijing Tong ren Tang drug store, whose origin is from Anqing city, Anhui province, China. Then, the medicinal herbs were identified by Professor Lin Ma according to their characters (School of Chinese Materia Medica, Tianjin University of Traditional Chinese Medicine). The voucher specimen was deposited at the Academy of Traditional Chinese Medicine of Tianjin University of TCM.

Extraction and Isolation
The dried peels of P. granatum (9.0 kg) were extracted three times with 70% EtOH under reflux for 3, 2 and 2 h, successively. Evaporation of the solvent under reduced pressure provided the 70% EtOH extract (2450.0 g). RAW 264.7 macrophages-like cells were seeded in 96-well plates (2 × 10 5 cells/mL) and incubated for 24 h. After that, the supernatant was removed and RAW 264.7 cells were treated without or with test compounds (their primary concentration was 100 mM, and these solutions were then diluted to be 30 µM for compounds 1-18 and 20 and 15 µM for compound 19 as final concentration, respectively) for 18 h, respectively. Then, the supernatant was replaced with 0.5 mg/mL of MTT solution and incubated further. After 4 h, the supernatant was discarded, and the pellet at the bottom of the 96-well plate was dissolved with DMSO, measured at 490 nm using a BioTek Cytation five-cell imaging multi-mode reader (Winooski, VT, USA).

Determination of NO Production
The in vitro NO production inhibitory assay was conducted as what has been reported [29]. In the experiment, four groups, including the normal group, LPS group, the positive drug DEX group and the tested groups, were settled. RAW 264.7 cells were seeded at the 96-well plates of 2 × 10 6 cells/mL and incubated for 24 h to facilitate complete adherence to the well. The primary compound solution was the same as that in Section 4.2.2, with a concentration at 100 mM, and then, these solutions were diluted to 3, 10 and 30 µM for compounds 1, 3, 5-8, 10, 11, 14, 16-18 and 20 and 1, 5 and 15 µM for compound 19, respectively.
Then, the media of the normal group was replaced with serum-containing medium, LPS (0.5 µg/mL) was administered to the LPS group, LPS (0.5 µg/mL) combined with positive drug DEX (1.5 µg/mL) was given to the DEX group, and the tested compounds together with LPS (0.5 µg/mL) were applied to the tested groups, followed by incubation 18 h. After, equal supernatant of the RAW 264.7 culture and the Griess reagent from Beyotime Biotechnology (Shanghai, China) were mixed and measured at 540 nm on a BioTek Cytation five-cell imaging multi-mode reader (Winooski, VT, USA). A standard sodium nitrite curve was used to calculate the amount of NO.

Western Blot Analysis
RAW 264.7 cells were seeded in the 6-cell plates for 24 h. Then, the supernatant of normal group, LPS group, the positive drug DEX group and the tested groups were replaced with serum-containing DMEM, LPS (0.5 µg/mL), LPS (0.5 µg/mL) as well as DEX (1.5 µg/mL) and LPS (0.5 µg/mL) combined with the tested compounds (final concentration was 30 µM), respectively. After 18 h, the RAW 264.7 cells were separated from the 6-cell plates (4 × 10 6 cells/mL) and then centrifuged at 4 • C and 12,000× g for 5 min. The cells were lysed by adding mixed solution of RIPA lysis buffer, protease and phosphatase inhibitor with the ratio of 100:1:1 for 30 min on ice.
Then, the supernatant was collected by centrifuging at 12,000× g for 5 min, and the total protein content was determined by using a BCA protein quantification kit (Thermo Fisher Scientific, Waltham, MA, USA). Next, the protein sample was mixed with 4 × sample buffer, incubated at 100 • C for 5 min, loaded onto 10% sodium dodecyl sulfate (SDS) polyacrylamide gel and electrophoresed. The electrophoretic SDS polyacrylamide gel was transferred onto polyvinylidene fluoride membranes (Merch/Millipore, Schwalbach, Germany).
Then, the membranes were washed three times, each for 10 min with Tris-buffered saline with 0.1% Tween 20 (TBST), followed by treatment with horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG) (1:10,000, SR238; Beijing Solarbio Science & Technology, Beijing, China) at room temperature for 1 h and then washed with TBST buffer three times for 10 min. Finally, the immunoreactive protein bands were measured by Immobilon Western Chemilumescent HRP Substrate (Millipore, Massachusetts, USA), visualized using a ChemiDoc MP Imaging System (Bio-Rad Laboratories, Hercules, USA) and analyzed using Image Lab software (Version 1.0, National Institutes of Health, Bethesda, MD, USA).

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/molecules27144622/s1, Supplementary data (the NMR and HRESIMS spectra of compounds 1-9, ECD spectra of compounds 3, 4, 6-8, 14, 16-20, cell viability assay, the raw data for western blot assays, and the physical and chemical data of compounds 10-20 are available in the Supporting Information.